Copper and Zinc containing superoxide dismutase (SOD1) plays a critical role in protecting cells from oxidative stress. Oxidative stress and the resulting cellular damage is linked to several human diseases including cancer, cardiovascular disease, and neurological degradation. For proper activity, SOD1 requires several post-translational modifications including zinc binding, insertion of the catalytic copper, and the formation of an intramolecular disulfide bond. However, dominate, toxic gain of function mutations in SOD1 can lead to amyotrophic lateral sclerosis (ALS), or Lou Gehrigs disease. While the underlying mechanism is still unclear, an emerging theme involves the misfolding and aggregation of SOD1 mutants. Metal binding and disulfide formation stabilize SOD1 and decrease aggregation. Therefore, understanding the molecular pathways of SOD1 maturation is relevant to human health. SOD1 maturation occurs through two pathways, a pathway dependent on the copper chaperone for SOD1 (CCS) and a second pathway independent of CCS. The molecular determinants and factors involved in the CCS independent pathway are relatively unknown. We hypothesize that the CCS independent pathway requires a SOD1 substrate with a high propensity of disulfide oxidation and specific cellular factors to deliver the catalytic copper. We will test this by: Aim 1 - To understand the role of the SOD1 disulfide in choice of copper activation pathways. We will use a combination of biochemical and yeast genetic techniques to test the role of the disulfide. We will measure the disulfide reduction potential of SOD1s that are CCS dependent, independent, or capable of using both pathways. We will also look at the role of molecular oxygen in CCS independent SOD1activation and disulfide bond oxidation. And, we will identify specific amino-acids of the SOD1 polypeptide that regulate activation pathway preference and disulfide oxidation propensity. Aim 2 - Determine genes required for CCS independent activation. We will screen for mutli-copy suppressors of a ccs??yeast strain by high throughput microarray analysis to identify genes involved in either copper delivery to SOD1 or genes involved in SOD1 disulfide oxidation. These studies will shed new light into the biology of SOD1 maturation with important implications to disease. Reactive oxygen causes cellular damage leading diseases such as cancer. This research proposal exploits the simple organism, bakers yeast, to understand the mechanisms of cellular defense to reactive oxygen.